CN116555137A - 一种苏氨酸生产菌株及其应用 - Google Patents
一种苏氨酸生产菌株及其应用 Download PDFInfo
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- CN116555137A CN116555137A CN202210114612.8A CN202210114612A CN116555137A CN 116555137 A CN116555137 A CN 116555137A CN 202210114612 A CN202210114612 A CN 202210114612A CN 116555137 A CN116555137 A CN 116555137A
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Abstract
本发明涉及微生物工程技术领域，具体涉及一种苏氨酸生产菌株及其应用。本发明通过构建转酮酶活性增强的菌株并将其应用于苏氨酸生产，显著提高了菌株的苏氨酸生产能力，苏氨酸产量较未经改造的菌株显著提高；结合6‑磷酸葡萄糖酸脱氢酶、NADP依赖性甘油醛3‑磷酸脱氢酶以及苏氨酸合成途径相关的酶的活性增强，苏氨酸的产量进一步提升。上述改造可应用于苏氨酸的发酵生产中，具有较好的应用价值。
Description
技术领域
本发明涉及微生物工程技术领域，具体涉及一种苏氨酸生产菌株及其应用。
背景技术
L-苏氨酸(L-Threonin)，化学名称为β-羟基-α-氨基丁酸，分子式为C4H9NO3，相对分子质量为119.12，为白色斜方晶系或结晶性粉末，无臭，味微甜，253℃熔化并分解，高温下溶于水，25℃溶解度为20.5g/100ml，等电点5.6，不溶于乙醇、乙醚和氯仿。
L-苏氨酸是一种必需氨基酸，主要用于医药、化学试剂、食品强化剂、饲料添加剂等方面。苏氨酸在饲料添加剂领域具有重要的应用且用量增长迅速。苏氨酸常被添加至未成年仔猪和家禽的饲料中，是猪饲料的第二限制氨基酸和家禽饲料的第三限制氨基酸。在配合饲料中添加L-苏氨酸，可以调整饲料的氨基酸平衡，促进禽畜生长，改善肉质，改善氨基酸消化率低的饲料的营养价值，降低饲料原料成本等因此在欧盟国家(主要是德国、比利时、丹麦等)和美洲国家，苏氨酸已广泛地应用于饲料行业。
谷氨酸棒状杆菌中，由草酰乙酸生成苏氨酸需要五步催化反应，其催化酶分别为天冬氨酸激酶(lysC编码)、天冬氨酸半醛脱氢酶(asd编码)、高丝氨酸脱氢酶(hom编码)、高丝氨酸激酶(thrB编码)以及苏氨酸合酶(thrC编码)。目前利用谷氨酸棒状杆菌生产苏氨酸的报道主要集中在对苏氨酸的合成代谢路径的改造，其中包括：抗反馈抑制的hom基因和lysC基因(Reinscheid D J,Eikmanns B J,Sahm H.Analysis of a Corynebacteriumglutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.[J].Journal of Bacteriology,1991,173(10):3228-3230；Eikmanns B J,Eggeling L,Sahm H.Molecular aspects of lysine,threonine,and isoleucine biosynthesis inCorynebacterium glutamicum.[J].Antonie Van Leeuwenhoek,1993,64(2):145-163.)；以及弱化苏氨酸利用途径中的编码基因glyA，同时过表达苏氨酸外运蛋白ThrE(Simic P,Willuhn J,Sahm H,et al.Identification of glyA(Encoding SerineHydroxymethyltransferase)and Its Use Together with the Exporter ThrE ToIncrease l-Threonine Accumulation by Corynebacterium glutamicum[J].Appliedand Environmental Microbiology,2002,68(7):3321-3327.)等。
为提高苏氨酸的发酵生产效率，降低生产成本，构建能够高效生产苏氨酸的生产菌株具有重要意义。
发明内容
本发明的目的是通过增强转酮酶的活性使菌株生产苏氨酸的能力得到提升，从而提供一种苏氨酸生产菌株及其应用。
苏氨酸合成过程中需要消耗还原力，但是，谷氨酸棒状杆菌中的还原力合成受到严格的调控，还原力的合成和消耗的平衡是维持谷氨酸棒状杆菌的正常生长和保证代谢产物生产的关键。本发明在苏氨酸的代谢工程研究中发现，与其它还原力合成相关基因的改造相比，强化菌株的转酮酶，能够更有效地提升苏氨酸合成过程中的还原力供应，显著提高菌株的苏氨酸合成能力。
为实现本发明的目的，第一方面，本发明提供一种修饰的棒状杆菌属微生物，所述微生物相比于未修饰的微生物，其转酮酶的活性增强，且所述微生物相比于未修饰的微生物具有增强的苏氨酸生产能力。
优选地，转酮酶在NCBI上的参考序列编号为NP_600788.1，或与其相似性为90％且具有同等功能的氨基酸序列。
以上所述的活性增强是由选自以下1)～6)，或任选的组合实现的：
1)通过导入具有所述酶的编码基因的质粒而增强；
2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
5)通过对酶的氨基酸序列进行改变而增强；
6)通过对编码酶的核苷酸序列进行改变而增强。
可以采用诱变、定点突变或同源重组的方法来增强转酮酶的活性。
优选地，通过以下方式增强转酮酶的活性：采用Psod启动子启动染色体上原始的转酮酶编码基因的转录，同时，在染色体上增加一个拷贝的由Psod启动子启动转录的转酮酶编码基因。
进一步地，所述微生物相比于未修饰的微生物，其6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶的活性增强和/或解除反馈抑制。
优选地，6-磷酸葡萄糖酸脱氢酶、NADP依赖性甘油醛3-磷酸脱氢酶在NCBI上的参考序列编号分别为NP_600669.1、FOB93_04945，或与其相似性为90％且具有同等功能的氨基酸序列。
进一步地，所述微生物相比于未修饰的微生物，其体内与苏氨酸合成途径相关的酶的活性增强和/或解除反馈抑制；其中，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶中的至少一种。
优选地，天冬氨酸激酶、高丝氨酸脱氢酶在NCBI上的参考序列编号分别为WP_003855724.1、WP_003855724.1，或与其相似性为90％且具有同等功能的氨基酸序列。
优选地，所述微生物为如下①～④中的任一种：
①转酮酶活性增强且天冬氨酸激酶和/或高丝氨酸脱氢酶活性增强和/或解除反馈抑制的微生物；
②转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶和/或6-磷酸葡萄糖酸脱氢酶活性增强和/或解除反馈抑制的微生物；
③转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物；
④转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶、6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物。
上述酶的活性增强是由选自以下1)～6)，或任选的组合实现的：
1)通过导入具有所述酶的编码基因的质粒而增强；
2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
5)通过对酶的氨基酸序列进行改变而增强；
6)通过对编码酶的核苷酸序列进行改变而增强。
优选地，所述酶活性的增强通过采用较基因的原始启动子活性更强的强启动子启动基因的转录实现，和/或，通过将基因的起始密码子突变为ATG实现。
所述强启动子优选为启动子PcspB、Psod或Ptuf。其中，启动子Psod、PcspB、Ptuf的核苷酸序列分别如SEQ ID NO.1、2、3所示。
优选地，天冬氨酸激酶的活性增强通过在其起始密码子前插入启动子Psod，并将其起始密码子由GTG突变为ATG实现。
高丝氨酸脱氢酶的活性增强通过将其原始启动子替换为启动子PcspB实现。
6-磷酸葡萄糖酸脱氢酶的活性增强通过在其起始密码子前插入启动子Psod实现。
NADP依赖性甘油醛3-磷酸脱氢酶优选为变异链球菌来源的NADP依赖性甘油醛3-磷酸脱氢酶，其表达优选以Ptuf作为启动子。
上述天冬氨酸激酶的解除反馈抑制通过将天冬氨酸激酶突变，使其发生T311I突变实现；高丝氨酸脱氢酶的解除反馈抑制通过将高丝氨酸脱氢酶突变，使其发生G378E突变实现。
优选地，本发明所述微生物为谷氨酸棒状杆菌(Corynebacterium glutamicum)。谷氨酸棒状杆菌包括ATCC13032、ATCC13870、ATCC13869、ATCC21799、ATCC21831、ATCC14067、ATCC13287等(参见NCBI Corunebacterium glutamicum进化树https://www.ncbi.nlm.nih.gov/genome/469)，更优选谷氨酸棒状杆菌ATCC 13032。
第二方面，本发明提供产苏氨酸菌株的构建方法，所述方法包括：
A、强化具有氨基酸生产能力的棒状杆菌中编码转酮酶的基因的表达，获得基因强化菌株；和/或
B、增强6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶的活性和/或将其解除反馈抑制；和/或
C、增强与苏氨酸合成途径相关的酶的活性和/或将其解除反馈抑制，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶中的至少一种；
所述活性增强的途径选自以下1)～6)，或任选的组合：
1)通过导入具有所述酶的编码基因的质粒而增强；
2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
5)通过对酶的氨基酸序列进行改变而增强；
6)通过对编码酶的核苷酸序列进行改变而增强。
第三方面，本发明提供一种生产苏氨酸的方法，所述方法包括如下步骤：
a)培养所述微生物，以获得所述微生物的培养物；
b)从步骤a)中获得的所述培养物中收集所产生的苏氨酸。
第四方面，本发明提供本发明提供转酮酶的活性增强在苏氨酸发酵生产或提高苏氨酸发酵产量中的应用。
进一步地，通过强化具有氨基酸生产能力的棒状杆菌(Corynebacterium)中的转酮酶的表达来提高苏氨酸的发酵产量。
优选地，本发明所述棒状杆菌为谷氨酸棒状杆菌(Corynebacteriumglutamicum)，谷氨酸棒状杆菌包括ATCC13032、ATCC13870、ATCC13869、ATCC21799、ATCC21831、ATCC14067、ATCC13287等(参见NCBI Corunebacterium glutamicum进化树https://www.ncbi.nlm.nih.gov/genome/469)，更优选谷氨酸棒状杆菌ATCC 13032。
第五方面，本发明提供所述修饰的棒状杆菌属微生物或按照上述方法构建得到的产苏氨酸菌株在苏氨酸发酵生产或提高苏氨酸发酵产量中的应用。
上述有关菌株的改造方法包括基因的强化和弱化等均为本领域技术人员可知的改造方式，参见满在伟.高产L-精氨酸钝齿棒杆菌的系统途径工程改造[D].江南大学,2016；崔毅.代谢工程改造谷氨酸棒杆菌生产L-亮氨酸[D].天津科技大学.；徐国栋.L-异亮氨酸生产菌株的构建及发酵条件优化.天津科技大学,2015.
本发明的有益效果在于：本发明通过对磷酸戊糖途径的转酮酶的强化表达，显著提高了苏氨酸合成过程中的还原力供应，进而提高了菌株的苏氨酸合成能力，苏氨酸产量较未经改造的菌株显著提高；结合6-磷酸葡萄糖酸脱氢酶、NADP依赖性甘油醛3-磷酸脱氢酶以及苏氨酸合成途径相关的酶的活性增强，苏氨酸的产量进一步提升。上述改造可应用于苏氨酸的发酵生产中，具有较好的应用价值。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
以下实施例中所涉及的酶和基因的详细信息如下：
转酮酶，编码基因名称tkt，NCBI编号：cg1774，Cgl1574，NCgl1512。
天冬氨酸激酶，编码基因名称lysC，NCBI编号：cg0306，Cgl0251，NCgl0247。
高丝氨酸脱氢酶，编码基因名称hom，NCBI编号：cg1337，Cgl1183，NCgl1136。
6-磷酸葡萄糖酸脱氢酶,编码基因名称gnd,NCBI编号：cg1643，Cgl1452，NCgl1396。
NADP依赖性甘油醛3-磷酸脱氢酶,编码基因名称gapN，NCBI编号：FOB93_04945。
实施例1菌株基因组改造质粒构建
1、转酮酶启动子强化表达质粒pK18mobsacB-Psod-tkt的构建
以ATCC13032基因组为模板，以P175/P176引物对进行PCR扩增得到上游同源臂up，以P177/P178引物对进行PCR扩增得到启动子片段Psod，以P179/P180引物对进行PCR扩增得到下游同源臂down，以P175/P180引物对以up、Psod、down为模板进行融合PCR，获得片段tkt-up-Psod-down。pK18mobsacB用BamHI/HindIII酶切。将酶切后的pK18mobsacB和tkt-up-Psod-down用无缝克隆试剂盒进行组装，转化Trans1 T1感受态细胞，获得重组质粒pK18mobsacB-Psod-tkt。
2、转酮酶启动子强化表达二拷贝质粒pK18mobsacB-Psod-tkt2nd的构建
质粒构建方法参考上述1，分别使用tkt-1f/1r,tkt-2f/2r,tkt-3f/3r,tkt-4f/4r四对引物扩增出4个小片段，使用tkt-1f/2r，tkt-3f/4r进行1次融合PCR，再使用tkt-1f/4r进行第二次融合PCR，获得全长片段。再按照上述1中的方法构建获得重组质粒pK18mobsacB-Psod-tkt2nd。利用该重组质粒可在染色体上插入一个额外拷贝的tkt基因，该tkt基因由Psod启动转录，Psod-tkt的核苷酸序列如SEQ ID NO.4所示。
3、天冬氨酸激酶表达强化质粒pK18mobsacB-Psod-lysCg1a-T311I的构建
质粒构建方法参考上述1，所用引物为P21-P28。
4、高丝氨酸脱氢酶表达强化质粒pK18mobsacB-PcspB-homG378E的构建
质粒构建方法参考上述1，所用引物为P29-P36。
5、6-磷酸葡萄糖酸脱氢酶表达强化质粒pK18mobsacB-Psod-gnd的构建
质粒构建方法参考上述1，所用引物为P123-P128。
6、NADP依赖性甘油醛3-磷酸脱氢酶表达强化质粒pK18mobsacB-Ptuf-gapN的构建
质粒构建方法参考上述1，gapN基因扩增的模板使用变异链球菌基因组，所用引物为P137-P144。利用pK18mobsacB-Ptuf-gapN质粒可在染色体上插入异源gapN基因。
以上质粒构建过程中所用的引物如表1所示。
表1引物序列
实施例2基因组改造菌株的构建
1、转酮酶启动子强化表达菌株的构建
按照谷氨酸棒状杆菌经典方法(C.glutamicum Handbook,Charpter 23)制备ATCC13032感受态细胞。重组质粒pK18mobsacB-Psod-tkt以电穿孔方法转化该感受态细胞，并在含有15mg/L卡那霉素的选择培养基上筛选转化子，其中目的基因由于同源性被插入到染色体中。将筛得的转化子过夜培养于普通液体脑心浸液培养基中，培养温度为30℃，回转摇床220rpm振荡培养。此培养过程中，转化子发生第二次重组，通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10-2连续稀释至10-4)，稀释液涂布在含有10％蔗糖的普通固体脑心浸液培养基上，33℃静置培养48h。蔗糖培养基上长出的菌落的基因组中不携带插入的载体序列。通过PCR扩增目的片段并进行核苷酸测序分析，获得目的突变菌株命名为SMCT151，该菌株与菌株ATCC13032相比，在tkt基因起始密码子TTG前插入Psod启动子。
2、转酮酶多拷贝强化表达菌株的构建
菌株构建方法参考上述1，以SMCT151为出发菌，转化pK18mobsacB-Psod-tkt2nd质粒，进行转酮酶二拷贝强化表达改造，获得的改造菌株命名为SMCT152，该菌株与菌株SMCT151相比，在染色体的pgl基因(cgl1578)终止密码子后增加了一个拷贝的由Psod启动转录的tkt基因。
3、天冬氨酸激酶和高丝氨酸脱氢酶强化表达菌株的构建
菌株构建方法参考上述1，以ATCC13032为出发菌，叠加转化pK18mobsacB-Psod-lysCg1a-T311I，pK18mobsacB-PcspB-homG378E质粒，进行天冬氨酸激酶和高丝氨酸脱氢酶的强化表达改造，获得的改造菌株命名为SMCT153，该菌株与ATCC13032菌株相比，lysC基因发生突变导致其起始密码子由GTG突变为ATG，编码氨基酸的第311位由苏氨酸突变为异亮氨酸，同时lysC基因起始密码子前插入Psod启动子，hom基因发生突变导致其编码蛋白产生G378E的突变，同时hom基因ATG前插入PcspB启动子。
4、6-磷酸葡萄糖酸脱氢酶表达强化菌株的构建
菌株构建方法参考上述1，以SMCT153为出发菌，转化pK18mobsacB-Psod-gnd质粒，进行6-磷酸葡萄糖酸脱氢酶强化表达改造，获得的改造菌株命名为SMCT154，该菌株与菌株SMCT153相比，gnd基因起始密码子ATG前插入Psod启动子。
5、NADP依赖性甘油醛3-磷酸脱氢酶表达强化菌株的构建
菌株构建方法参考上述1，分别以SMCT153，SMCT154为出发菌，转化pK18mobsacB-Ptuf-gapN质粒，进行NADP依赖性甘油醛3-磷酸脱氢酶强化表达改造，获得的改造菌株分别命名为SMCT155，SMCT156，这两株菌与其对应的出发菌相比，在染色体的cgl1705基因的终止密码子之后插入了由Ptuf启动转录的gapN基因。
6、转酮酶多拷贝强化表达叠加菌株构建
分别以SMCT153，SMCT154，SMCT155，SMCT156为出发菌，叠加转化pK18mobsacB-Psod-tkt和pK18mobsacB-Psod-tkt2nd，改造获得的菌株分别命名为SMCT157，SMCT158，SMCT159，SMCT160，这些菌株与其对应的出发菌相比，在tkt基因的起始密码子TTG前插入Psod启动子，同时，在染色体的pgl基因终止密码子之后增加了一个拷贝的由Psod启动转录的tkt基因。
上述获得的菌株基因型信息如表2所示。
表2菌株基因型信息
实施例3菌株的摇瓶发酵验证
对实施例2构建的各改造菌株进行摇瓶发酵验证，具体如下：
1、培养基
种子活化培养基：BHI 3.7％，琼脂2％，pH 7。
种子培养基：蛋白胨5/L，酵母抽提物5g/L，氯化钠10g/L，硫酸铵16g/L，尿素8g/L，磷酸二氢钾10.4g/L，磷酸氢二钾21.4g/L，生物素5mg/L，硫酸镁3g/L。葡萄糖50g/L，pH7.2。
发酵培养基：玉米浆50mL/L，葡萄糖30g/L，硫酸铵4g/L，MOPS 30g/L，磷酸二氢钾10g/L，尿素20g/L，生物素10mg/L，硫酸镁6g/L，硫酸亚铁1g/L，VB1·HCl 40mg/L，泛酸钙50mg/L，烟酰胺40mg/L，硫酸锰1g/L，硫酸锌20mg/L，硫酸铜20mg/L，pH 7.2。
2、工程菌摇瓶发酵生产L-苏氨酸
(1)种子培养：挑取SMCT151、SMCT152、SMCT153、SMCT154、SMCT155、SMCT156、SMCT157、SMCT158、SMCT159、SMCT160的斜面种子1环接至装有20mL种子培养基的500mL三角瓶中，于30℃、220r/min振荡培养16h，得到种子液。
(2)发酵培养：将2mL种子液接种至装有20mL发酵培养基的500mL三角瓶中，于33℃、220r/min振荡培养24h，得到发酵液。
(3)取1mL发酵液离心(12000rpm，2min)，收集上清液，用HPLC检测工程菌与对照菌发酵液中的L-苏氨酸。
苏氨酸的摇瓶发酵结果如表3所示。
表3发酵检测结果
结果显示，在SMCT153、SMCT154、SMCT155、SMCT156中强化tkt基因的表达均使得菌株的苏氨酸产量显著提高，其中，在SMCT156菌株中强化tkt基因的表达后，菌株的苏氨酸产量提升了5g/L，表明增强苏氨酸合成过程中所需还原力的供应可明显提升菌株生产苏氨酸的能力，而将提升还原力供应的改造与苏氨酸末端合成路径相关基因的表达强化组合，菌株生产苏氨酸产量会进一步提升。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 廊坊梅花生物技术开发有限公司
<120> 一种苏氨酸生产菌株及其应用
<130> KHP211124135.0
<160> 47
<170> SIPOSequenceListing 1.0
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tagctgccaa ttattccggg cttgtgaccc gctacccgat aaataggtcg gctgaaaaat 60
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gaagcgccat ctgacggatt ttcaaaagat gtatatgctc ggtgcggaaa cctacgaaag 180
gattttttac cc 192
<210> 2
<211> 260
<212> DNA
<213> 人工序列(Artificial Sequence)
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acctgcgttt ataaagaaat gtaaacgtga tcggatcgat ataaaagaaa cagtttgtac 60
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tttctcaacg acaggctgct aagctgctag ttcggtggcc tagtgagtgg cgtttacttg 180
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<210> 3
<211> 200
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tggccgttac cctgcgaatg tccacagggt agctggtagt ttgaaaatca acgccgttgc 60
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<210> 4
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tagctgccaa ttattccggg cttgtgaccc gctacccgat aaataggtcg gctgaaaaat 60
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gaagcgccat ctgacggatt ttcaaaagat gtatatgctc ggtgcggaaa cctacgaaag 180
gattttttac ccttgaccac cttgacgctg tcacctgaac ttcaggcgct cactgtacgc 240
aattacccct ctgattggtc cgatgtggac accaaggctg tagacactgt tcgtgtcctc 300
gctgcagacg ctgtagaaaa ctgtggctcc ggccacccag gcaccgcaat gagcctggct 360
ccccttgcat acaccttgta ccagcgggtt atgaacgtag atccacagga caccaactgg 420
gcaggccgtg accgcttcgt tctttcttgt ggccactcct ctttgaccca gtacatccag 480
ctttacttgg gtggattcgg ccttgagatg gatgacctga aggctctgcg cacctgggat 540
tccttgaccc caggacaccc tgagtaccgc cacaccaagg gcgttgagat caccactggc 600
cctcttggcc agggtcttgc atctgcagtt ggtatggcca tggctgctcg tcgtgagcgt 660
ggcctattcg acccaaccgc tgctgagggc gaatccccat tcgaccacca catctacgtc 720
attgcttctg atggtgacct gcaggaaggt gtcacctctg aggcatcctc catcgctggc 780
acccagcagc tgggcaacct catcgtgttc tgggatgaca accgcatctc catcgaagac 840
aacactgaga tcgctttcaa cgaggacgtt gttgctcgtt acaaggctta cggctggcag 900
accattgagg ttgaggctgg cgaggacgtt gcagcaatcg aagctgcagt ggctgaggct 960
aagaaggaca ccaagcgacc taccttcatc cgcgttcgca ccatcatcgg cttcccagct 1020
ccaactatga tgaacaccgg tgctgtgcac ggtgctgctc ttggcgcagc tgaggttgca 1080
gcaaccaaga ctgagcttgg attcgatcct gaggctcact tcgcgatcga cgatgaggtt 1140
atcgctcaca cccgctccct cgcagagcgc gctgcacaga agaaggctgc atggcaggtc 1200
aagttcgatg agtgggcagc tgccaaccct gagaacaagg ctctgttcga tcgcctgaac 1260
tcccgtgagc ttccagcggg ctacgctgac gagctcccaa catgggatgc agatgagaag 1320
ggcgtcgcaa ctcgtaaggc ttccgaggct gcacttcagg cactgggcaa gacccttcct 1380
gagctgtggg gcggttccgc tgacctcgca ggttccaaca acaccgtgat caagggctcc 1440
ccttccttcg gccctgagtc catctccacc gagacctggt ctgctgagcc ttacggccgt 1500
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cacggtggca cccgcccata cggcggaacc ttcctcatct tctccgacta catgcgtcct 1620
gcagttcgtc ttgcagctct catggagacc gacgcttact acgtctggac ccacgactcc 1680
atcggtctgg gcgaagatgg cccaacccac cagcctgttg aaaccttggc tgcactgcgc 1740
gccatcccag gtctgtccgt cctgcgtcct gcagatgcga acgagaccgc ccaggcttgg 1800
gctgcagcac ttgagtacaa ggaaggccct aagggtcttg cactgacccg ccagaacgtt 1860
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<210> 5
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 6
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<210> 10
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<212> DNA
<213> 人工序列(Artificial Sequence)
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acgacggcca gtgccaagct ttagatgtgg tggtcgaatg gg 42
<210> 11
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<212> DNA
<213> 人工序列(Artificial Sequence)
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aattcgagct cggtacccgg ggatccagcg acaggacaag cactgg 46
<210> 12
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
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cccggaataa ttggcagcta tgtgcacctt tcgatctacg 40
<210> 13
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
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cgtagatcga aaggtgcaca tagctgccaa ttattccggg 40
<210> 14
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<212> DNA
<213> 人工序列(Artificial Sequence)
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tttctgtacg accagggcca tgggtaaaaa atcctttcgt a 41
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<212> DNA
<213> 人工序列(Artificial Sequence)
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tcggaacgag ggcaggtgaa ggtgatgtcg gtggtgccgt ct 42
<210> 16
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<212> DNA
<213> 人工序列(Artificial Sequence)
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agacggcacc accgacatca ccttcacctg ccctcgttcc ga 42
<210> 17
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gtaaaacgac ggccagtgcc aagcttagcc tggtaagagg aaacgt 46
<210> 18
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<212> DNA
<213> 人工序列(Artificial Sequence)
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aattcgagct cggtacccgg ggatccctgc gggcagatcc ttttga 46
<210> 19
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
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atttctttat aaacgcaggt catatctacc aaaactacgc 40
<210> 20
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gcgtagtttt ggtagatatg acctgcgttt ataaagaaat 40
<210> 21
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gtatatctcc ttctgcagga ataggtatcg aaagacgaaa 40
<210> 22
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tttcgtcttt cgatacctat tcctgcagaa ggagatatac 40
<210> 23
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
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tagccaattc agccaaaacc cccacgcgat cttccacatc c 41
<210> 24
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ggatgtggaa gatcgcgtgg gggttttggc tgaattggct a 41
<210> 25
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gtaaaacgac ggccagtgcc aagcttgctg gctcttgccg tcgata 46
<210> 26
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
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<210> 27
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ggaaacctac gaaaggattt tttacccatg actaatggag ataatctcgc acag 54
<210> 28
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ctgtgcgaga ttatctccat tagtcatggg taaaaaatcc tttcgtaggt ttcc 54
<210> 29
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
gtaaaatcgc cactaccccc aaatggttag ctgccaatta ttccgggctt gtga 54
<210> 30
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
tcacaagccc ggaataattg gcagctaacc atttgggggt agtggcgatt ttac 54
<210> 31
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gttgtaaaac gacggccagt gccaagcttc atggtgcgca gtgtggttcg tgcgacg 57
<210> 32
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
acgaattcga gctcggtacc cggggatcct gtttacctga cactcaagcc ccgtgcac 58
<210> 33
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
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<210> 34
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
accactgacc cagattatct cagactaagc ggcttgttag atatcttgaa atcggc 56
<210> 35
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
ttgacataat ttttatattg ttttgtcatt tactgaatcc taagggcaac ggcgttga 58
<210> 36
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
tcaacgccgt tgcccttagg attcagtaaa tgacaaaaca atataaaaat tatgtcaa 58
<210> 37
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
agatgaagta ggtgggtgaa tatagctgtt atttgatatc aaatacgacg gattta 56
<210> 38
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
taaatccgtc gtatttgata tcaaataaca gctatattca cccacctact tcatct 56
<210> 39
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ttgtaaaacg acggccagtg ccaagcttga ttggaatcgg catgggtgtt ctgcgt 56
<210> 40
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gagctcggta cccggggatc cgatgagcgc aatgtccctg t 41
<210> 41
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
cccggaataa ttggcagcta ttagagattt cctgcagcat ca 42
<210> 42
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
atgatgctgc aggaaatctc taatagctgc caattattcc gggcttgt 48
<210> 43
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
caggtgacag cgtcaaggtg gtcaagggta aaaaatcctt tcgtagg 47
<210> 44
<211> 47
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
cctacgaaag gattttttac ccttgaccac cttgacgctg tcacctg 47
<210> 45
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
cttcttgtta gagctggcgc tgcttaaccg ttaatggagt ccttggccgc 50
<210> 46
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
gcggccaagg actccattaa cggttaagca gcgccagctc taacaagaag 50
<210> 47
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
acgacggcca gtgccaagct tgtggatcca ggttaagcgc a 41
Claims (10)
1.一种修饰的棒状杆菌属微生物，其特征在于，所述微生物相比于未修饰的微生物，其转酮酶的活性增强，且所述微生物相比于未修饰的微生物具有增强的苏氨酸生产能力。
2.根据权利要求1所述的微生物，其特征在于，所述活性增强是由选自以下1)～6)，或任选的组合实现的：
1)通过导入具有所述酶的编码基因的质粒而增强；
2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
5)通过对酶的氨基酸序列进行改变而增强；
6)通过对编码酶的核苷酸序列进行改变而增强。
3.根据权利要求2所述的微生物，其特征在于，采用诱变、定点突变或同源重组的方法来增强转酮酶的活性。
4.根据权利要求1所述的微生物，其特征在于，所述微生物相比于未修饰的微生物，其6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶的活性增强和/或解除反馈抑制。
5.根据权利要求1～4任一项所述的微生物，其特征在于，所述微生物相比于未修饰的微生物，其体内与苏氨酸合成途径相关的酶的活性增强和/或解除反馈抑制；
其中，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶中的至少一种。
6.根据权利要求5所述的微生物，其特征在于，所述微生物为如下①～④中的任一种：
①转酮酶活性增强且天冬氨酸激酶和/或高丝氨酸脱氢酶活性增强和/或解除反馈抑制的微生物；
②转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶和/或6-磷酸葡萄糖酸脱氢酶活性增强和/或解除反馈抑制的微生物；
③转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物；
④转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶、6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物。
7.根据权利要求4或5所述的微生物，其特征在于，所述活性增强是由选自以下1)～6)，或任选的组合实现的：
1)通过导入具有所述酶的编码基因的质粒而增强；
2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
5)通过对酶的氨基酸序列进行改变而增强；
6)通过对编码酶的核苷酸序列进行改变而增强。
8.根据权利要求1-7任一项所述的微生物，其特征在于，所述微生物为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
9.产苏氨酸菌株的构建方法，其特征在于，所述方法包括：
A、强化具有氨基酸生产能力的棒状杆菌中编码转酮酶的基因的表达，获得基因强化菌株；和/或
B、增强6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶的活性和/或将其解除反馈抑制；和/或
C、增强与苏氨酸合成途径相关的酶的活性和/或将其解除反馈抑制，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶中的至少一种；
所述活性增强的途径选自以下1)～6)，或任选的组合：
1)通过导入具有所述酶的编码基因的质粒而增强；
2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
5)通过对酶的氨基酸序列进行改变而增强；
6)通过对编码酶的核苷酸序列进行改变而增强。
10.一种生产苏氨酸的方法，其特征在于，所述方法包括如下步骤：
a)培养权利要求1-8任一项所述的微生物，以获得所述微生物的培养物；
b)从步骤a)中获得的所述培养物中收集所产生的苏氨酸。
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| AU5982200A (en) * | 2000-03-17 | 2001-09-24 | Degussa A.G. | Process for the fermentative preparation of l-amino acids with amplification of the tkt gene |
| US20030109014A1 (en) * | 2000-03-17 | 2003-06-12 | Kevin Burke | Process for the fermentative preparation of L-amino acids with amplification of the tkt gene |
| RU2288264C2 (ru) * | 2004-02-12 | 2006-11-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) | Бактерия, принадлежащая к роду escherichia - продуцент l-треонина и способ получения l-треонина |
| CN113322218B (zh) * | 2020-02-28 | 2022-11-22 | 廊坊梅花生物技术开发有限公司 | 重组谷氨酸棒杆菌及生产l-苏氨酸的方法 |
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